High-efficiency, high throughput chromatography has greatly advanced the analysis of complex chemical and biological mixtures. The ability of a chromatographic column to separate the various components present in a complex multi-component mixture heavily depends on the efficiency of the column. Column efficiency, in turn, is largely controlled by particle size of the material forming the chromatographic bed. The lower the particle size, the higher the column efficiency. As a result, with time, the particle size for chromatographic separation has become progressively smaller.
An important factor for achieving higher throughput chromatographic separation is short run time. This requires the mobile phase to be passed through the column at a high flow rate. The combination of low particle size and high flow rate leads to significant pressure drop in the mobile phase throughout the length of the chromatographic column. The extent of the pressure drop depends on the viscosity of the mobile phase. Pressure drop in the mobile phase requires use of pumps that can push the mobile phase into the column with greater and greater force. As a result, chromatographic instruments have continuously evolved to incorporate both smaller particles and high flow rates as exemplified by the development of Ultra High Performance Liquid Chromatography (or UHPLC) instruments that are capable of forcing the mobile phase at much higher flow rates compared to the traditional High Performance Liquid Chromatography (HPLC) instruments.
Pressure drop in the mobile phase is accompanied with a rise in the temperature of the mobile phase due to increased friction between the mobile phase and the particles of the chromatographic bed. The larger the pressure drop, the greater the extent of temperature rise. In most situations neither the mobile phase nor the chromatographic bed is sufficiently thermally conductive to equilibrate the temperature rise uniformly from the interior of the column to the areas closest to the column wall. The mobile phase near the wall can dissipate the frictional heat more easily through the wall and therefore can maintain a temperature closer to the temperature set by a column oven. If the column is not in contact with a heater (i.e., it is kept in the open under ambient temperature), it maintains a temperature closer to the ambient temperature. However, the mobile phase along the column central axis (i.e., lengthwise along center of column) fails to reach the temperature of the mobile phase near the wall. The temperature difference results in a radial thermal gradient. A thermal gradient in the radial direction, in general, reduces column efficiency due to band broadening.
Similar thermal effects are also observed in CO2-based chromatography when high flow rates and lower particle sizes are employed. However, the immediate physical consequences that ultimately lead to band broadening in CO2-based chromatography are different from those observed in UHPLC. In CO2-based chromatography, because of the significant concentration of CO2 in the mobile phase, the viscosity of the mobile phase is much lower. Lower viscosity considered alone should result in a smaller pressure drop, and therefore, less temperature variation in the radial direction in comparison to UHPLC. However, unlike UHPLC, pressure drop in CO2-based chromatography leads to significant mobile phase expansion, which in turn produces a cooling effect. The net result is that the temperature of the mobile phase near the wall is higher than that along central axis of the column.
Although it is well known that operation of both UHPLC and CO2-based chromatography is associated with temperature variation and resultant loss in column performance, the extent to which the temperature varies is not readily estimated by current methods. In this regard, CO2-based chromatography is more complex compared to UHPLC, because in CO2-based chromatography the physical properties of the mobile phase can vary significantly as a function of the operating pressure and temperature (as compared to liquids in UHPLC, due to significant changes in density). Consequently widely different temperature variations may be experienced along the column during CO2-based chromatography, depending on the selection of operating pressure and temperature.
Temperature variation aspects of both UHPLC and CO2-based chromatography systems have been examined experimentally and through simulation studies. For example, see Kaczmarski, et al., (2009) J. Chromatogr. A, 1216 (38), pp. 6575-6586, and Kaczmarski, K., (2010) J. of Chromatogr. A, 1217 (42), pp. 6578-6587, which are incorporated herein by reference in their entireties. Although these studies have generated useful insight into the nature of the temperature variation, the methods employed to estimate the variation have been complex, and therefore, not helpful in most practical situations.